Oral Presentation International Plant Molecular Biology Conference 2024

NMR, UPLC-MS and XL-MS to characterise enzymatic activity of plant TIR domains in innate immunity  (#345)

Mitchell Sorbello 1 , Bryan Lim 1 , Natsumi Maruta 1 , Mehdi Mobli 2 , Bostjan Kobe 1
  1. School of Chemistry and Molecular Biosciences and Australian Infection Diseases Research Centre, University of Queensland, Brisbane, QLD, Australia
  2. Centre for Advanced Imaging, Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St Lucia , QLD, Australia

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) detect virulence factors (effector proteins) that are secreted by pathogens into plant cells. NLRs consist of two regulatory domains that mediate effector recognition and NLR oligomerisation, and a variable N-terminal signalling domain. NLRs containing the N-terminal Toll-interleukin-1 receptor (TIR) domain (TNLs) possess NAD+ hydrolysis activity. Hydrolysis of NAD+ leads to production of a variety of compounds which activate downstream immune signalling pathways mediated by the enhanced disease susceptibility 1 (EDS1) family proteins. Currently, it is unknown whether all TNLs produce the same set of compounds to initiate signalling. Additionally, unpublished data suggests that TIR domains interact with the EDS1 family members. In this work, we aim to investigate the variability of plant TIR enzymatic activity and resulting products, using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR). We also aim to detect potential TIR:EDS1 interactions in vitro using cross-linking mass spectrometry (XL-MS) and electron microscopy. Optimisation of the XL-MS protocol is currently ongoing; our preliminary results indicate that plant TIRs produce different compounds upon NADase activity, highlighting alternate catalytic mechanisms or signalling pathways within the cell.